Methanotrophs are a group of ubiquitous Gram-negative bacteria that utilize methane as their sole source of carbon and energy. There are two enzymes used by methanotrophs to oxidize methane. One enzyme, the particulate methane monooxygenase (pMMO) is found in most known methanotrophs and is located in the cytoplasmic membrane. The other enzyme, the soluble methane monooxygenase (sMMO) is found is some methanotrophs and is located in the cytoplasm. Methanotrophic physiology is strongly affected by the amount of bioavailable copper. In methanotrophs that have both MMOs, copper is known to cause a shift in expression from the sMMO at low copper-to-biomass ratios to pMMO at high copper-to-biomass ratios. Additionally, the amount of pMMO produced increases exponentially with the amount of copper present.
Methylosinus trichosporium OB3b is thought to acquire copper by producing a copper chelating chalkophore, methanobactin. Methanobactin (mb) is a low molecular mass (1,154 Da) 7 amino acid chromopeptide observed in both the extracellular and membrane fraction of many, and perhaps all aerobic methanotrophs (DiSpirito, 2004; DiSpirito, 1998; Kim et al., 2004; Tellez et al., 1998; Zahn et al., 1996), which has very high affinity for copper.
When isolated from the membrane fraction, methanobactin contains one copper atom and is predominately associated with pMMO (Zahn et al., 1996; Choi et al., 2005; Choi et al., 2003). In the extracellular fraction, the majority of methanobactin is metal free (DiSpirito et al., 1998; Zahn et al., 1996). This proposed copper-siderophore, or chalkophore role (Kim et al., 2004), is based on copper uptake and localization studies (DiSpirito et al., 1998; Tellez et al., 1998; Zahn et al., 1996; Choi et al., 2005; Morton et al., 2000), chelation of copper in soil systems (Morton et al., 2000), characterization of constitutive sMMO mutants in Methylosinus trichosporium OB3b (DiSpirito et al., 1998; Tellez et al., 1998; Fitch et al., 1993; Phelps et al., 1992), and copper-binding studies (DiSpirito et al., Zahn et al., 1996; Choi et al., 2005; Choi et al., 2006; Kim et al., 2005).
The structure of copper containing methanobactin (Cu-methanobactin) following exposure to high copper concentrations shows one methanobactin binds one copper atom in a novel S, and N coordination by the 4-thiocarbonyl-5-hydroxy imidazolate (THI) and 4-hydroxy-5-thiocarbonyl imidazolate (HTI) moieties (Kim et al., 2004). However, spectral, kinetic and thermodynamic studies indicate that initial coordination of Cu(II) and Cu(I) differs from the coordination observed in the crystal structure (Choi et al., 2006). Methanobactin (mb) appears to initially coordinate Cu(II) as tetramer or oligomer by THI and possibly Tyrosine (FIG. 1, reaction 1). This initial coordination is followed by a reduction of Cu(II) to Cu(I) (FIG. 1, reaction 2), and then followed by a change in metal ligation, resulting in coordination by both THI and the HTI (FIG. 1, reaction 3). At Cu(II) to mb ratios above 0.25 the Cu(II) is coordinated as a dimer (FIG. 1, reaction 4), followed by coordination as a monomer at Cu(II) to mb ratios above 0.5 Cu per mb (FIG. 1, reactions 5, 6 and 7).
What is needed is a metal binding system useful in multiple applications.